Hypoallergenic allergy vaccines based on the timothy grass pollen allergen phl p7

ABSTRACT

The present invention pertains to polypeptides derived from the timothy grass pollen allergen Phi p 7. The polypeptides display reduced allergen activity and are useful as allergy vaccines for treatment of sensitized allergic patients and for prophylactic vaccination.

The present invention pertains to polypeptides derived from the timothygrass pollen allergen Phi p 7. The polypeptides display reducedallergenic activity and are useful as allergy vaccines for treatment ofsensitized allergic patients and for prophylactic vaccination.

More than 25% of the population suffers from IgE-mediated allergies. Thesymptoms of allergy (allergic rhinoconjunctivitis, asthma, dermatitis,anaphylactic shock) are due to IgE recognition of allergens. In order toinduce strong effector cell activation and thus inflammatory responses,an allergen must be able to cross-link effector cell-bound IgEantibodies efficiently. This process requires the presence of at leasttwo IgE epitopes on the allergen surface. IgE antibodies of allergicpatients may recognize either “continuous epitopes” consisting of a rowof consecutive amino acids or “discontinuous epitopes” which arecomposed of amino acids from different portions of the allergen broughtinto proximity by the molecule fold. Allergen-induced cross-linking ofmast cell-bound IgE antibodies induces the immediate release ofbiologically active mediators (e.g., histamine, leukotriens) whereasIgE-mediated presentation of allergens to T cells causes T cellactivation and release of proinflammatory cytokines.

About 5-20% of pollen-allergic individuals are sensitized to a recentlydiscovered group of calcium-binding proteins containing two bindingsites for calcium. These pollen allergens are therefore termed two-EFhand pollen allergens. Due to sequence similarities these proteinscontain cross-reactive IgE epitopes and sensitized patients showtherefore allergic symptoms upon contact with a great variety ofdifferent pollens from trees, grasses and weeds.

Allergen-specific immunotherapy currently represents one of the fewcurative forms of therapy for IgE-mediated allergies. It is currentlyconducted by the administration of allergen-containing extracts tosensitized individuals, mostly by injection but also via other routes(e.g., sublingual immunotherapy). While pollen extracts from trees,grasses and weeds are standardized regarding certain major allergens, nostandardization is performed regarding less frequently recognized buthighly cross-reactive allergens, e.g., the above mentionedcalcium-binding allergens.

Allergy to the two-EF hand pollen allergens can therefore not besufficiently treated with crude allergen extracts. Furthermore, two-EFhand allergens are highly allergenic and upon injection may inducesevere allergic side effects.

The invention aims at providing means for the prophylactic ortherapeutic treatment of allergy to two-EF hand pollen allergens. It hasbeen found that mutants of the timothy grass pollen allergen Phi p 7show strongly reduced IgE binding and are thus useful as hypoallergenicagents. The amino acid sequence of Phi p 7 is shown in SEQ ID NO:1. Theinvention relates to a mutated polypeptide derived from the pollenallergen Phi p 7 selected from the group consisting of

-   -   (a) polypeptides comprising an amino acid sequence in which in        respect to the amino acid sequence as shown in SEQ ID NO:1 one        to 15 amino acid residues are deleted, substituted and/or added;    -   (b) polypeptides comprising a fragment of (a), wherein the        fragment has a length of at least 15 amino acids and at least        90% of the amino acid residues of the fragment are identical to        corresponding residues of the amino acid sequence as shown in        SEQ ID NO:1;    -   (c) polypeptides comprising a fragment of the amino acid        sequence as shown in SEQ ID NO:1, wherein the fragment has a        length of at least 15 amino acids;    -   (d) polypeptides consisting of a fragment of (a), wherein the        fragment has a length of at least 10 amino acids and at least        80% of the amino acid residues of the fragment are identical to        corresponding residues of the amino acid sequence as shown in        SEQ ID NO:1; and    -   (e) polypeptides consisting of a fragment of the amino acid        sequence as shown in SEQ ID NO:1, wherein the fragment has a        length of at least 10 amino acids;        wherein the mutated polypeptide has reduced IgE binding activity        compared to wild type Phi p 7.

As used herein, the term “polypeptide” denotes a compound comprising atleast 7 amino acids which are linked by peptide bonds. The polypeptideis preferably composed only of amino acids, but it may also comprisenon-proteinaceous components. The length of the polypeptide ispreferably at least 10 amino acids, more preferably at least 15 aminoacids. The polypeptide may also be a fusion protein comprising a portionwhich is derived from Phi p 7 and a fusion partner. The portion derivedfrom Phi p 7 may further be linked to a carrier molecule, e.g. keyholelimpet hemocyanin (KLH).

The term “two-EF hand pollen allergen” as used herein designates acalcium binding polypeptide, the amino acid sequence of which is atleast 60% identical to the amino acid sequence as shown in SEQ ID NO:1.These polypeptides have amino acid sequences identical to the respectivenaturally occurring pollen allergens.

A “mutated” polypeptide according to the invention is a polypeptide theamino acid sequence of which is different from that of wild type ornaturally occurring two-EF hand pollen allergens.

An amino acid substitution denotes the replacement of one amino acidwith a different amino acid. Preferably, acidic residues aresubstituted. The substituting amino acid may be of any type, preferablyit is a non-acidic amino acid, more preferably a hydrophobic amino acidsuch as alanine, valine, leucine, etc.

In one embodiment, the polypeptide of the invention comprises an aminoacid sequence which has 1 to 15 amino acid deletions, substitutionsand/or additions in respect to the amino acid sequence as shown in SEQID NO:1. Preferably, the number of amino acid deletions, substitutionsand/or additions is 1 to 10, more preferably 1 to 6, most preferably 1to 4. The polypeptides of this embodiment are at least 63 amino acids inlength, the preferred length is about 78 amino acids. The preferredpolypeptides have 1, 2, 3 or 4 amino acid substitutions in respect tothe amino acid sequence as shown in SEQ ID NO:1. More preferably thepolypeptide comprises an amino acid sequence in which in respect to theamino acid sequence as shown in SEQ ID NO:1 the amino acids at position24 and 59, more preferably at position 17, 24 and 59, even morepreferably at position 17, 24, 52 and 59 are substituted. The mostpreferred polypeptides comprise an amino acid sequence as shown in SEQID NO:4, SEQ ID NO:5 or SEQ ID NO:6. The polypeptide represented by SEQID NO:4 carries two mutations as compared with the amino acid sequenceas shown in SEQ ID NO:1, namely E24A and D59A. The polypeptiderepresented by SEQ ID NO:5 has the mutations D17A, E24A, D59A ascompared with SEQ ID NO:1. The polypeptide represented by SEQ ID NO:6carries mutations D17A, E24A, D52A and E59A as compared with SEQ IDNO:1.

The invention further concerns polypeptides comprising a fragment of thepolypeptides described above (a). The fragment has a length of at least15 amino acids, i.e. it consists of at least 15 consecutive amino acidsof the polypeptide described above (a). Preferably, the length of thefragment is at least 20 amino acids, more preferably at least 25 aminoacids, even more preferably at least 30 amino acids. At least 90% of theamino acid residues of the fragment are identical to correspondingresidues of the amino acid sequence as shown in SEQ ID NO:1, preferablyat least 92%, most preferably at least 95%. Percent sequence identity isdetermined by conventional methods. The degree of identity of the aminoacid sequence of the fragment to SEQ ID NO:1 may be determined bycomparing the amino acid sequence of the fragment and SEQ ID NO:1 usingthe program “Blast 2 sequences version Blast p2.1.2” (Tatusova et al.(1999) FEMS Microbiol. Lett. 174, 247-250). The parameters which areused in this context are: matrix: BLOSUM 62; gap open: 11; gapextension: 1; X drop off: 50; expect: 10; word size: 3; filter: no.

In another embodiment, the polypeptide of the invention comprises afragment of the amino acid sequence as shown in SEQ ID NO:1 with alength of at least 15 amino acids. The fragment consists of at least 15consecutive amino acids of the amino acid sequence as shown in SEQ IDNO:1, preferably at least 20 consecutive amino acids, more preferably atleast 25 consecutive amino acids, most preferably at least 30consecutive amino acids.

The polypeptides of the invention may also consist of a fragment of thepolypeptides described above (a). This fragment consists of at least 10consecutive amino acids of the polypeptide described above (a),preferably at least 15 consecutive amino acids, more preferably at least20 consecutive amino acids, even more preferably at least 25 consecutiveamino acids, most preferably at least 30 consecutive amino acids. Theamino acid sequence of the fragment is at least 80% identical tocorresponding residues of the amino acid sequence as shown in SEQ IDNO:1. The degree of amino acid sequence identity is determined asdescribed supra. The sequence identity of the fragments to the aminoacid sequence of SEQ ID NO:1 is preferably at least 85%, more preferablyat least 90%, most preferably at least 95%.

In another embodiment, the polypeptide of the invention consists of afragment of the amino acid sequence as shown in SEQ ID NO:1. Thisfragment consists of at least 10 consecutive amino acids of the aminoacid sequence as shown in SEQ ID NO:1, preferably at least 15consecutive amino acids, more preferably at least 20 consecutive aminoacids, even more preferably 25 consecutive amino acids, most preferablyat least 30 consecutive amino acids. Preferred polypeptides comprise atleast the amino acids forming one of the EF hand motifs (amino acidsD13-L25 or D48-F60 of the amino acid sequence as shown in SEQ ID NO:1).Examples are the polypeptides consisting substantially of the amino acidsequence as shown in SEQ ID NO:2 (first EF hand motif or SEQ ID NO:3(second EF hand motif, respectively, optionally coupled to a carriermolecule such as KLH.

In accordance with the present invention, surface-exposed amino acidswhich potentially are involved in epitope formation may be substitutedor deleted. It has been found that the highly conserved residues lysine19 and phenylalanine 54 of SEQ ID NO:1 are solvent-exposed. Therefore,substitution or deletion of these residues may be carried out to obtaina polypeptide with reduced allergenic activity. Suggested mutations arethe residues asparagine 15, glycine 16 and aspartic acid 17 of SEQ IDNO:1. These residues form a kind of uncharged cap on top of theN-terminal calcium-binding loop. Substitution of these residues (e.g.with charged amino acids) will result in a completely different surfacepattern of this immunologically interesting protein region. Anothertarget is the mutation of the residues aspartic acid 50, aspartic acid52 and aspartic acid 56 of SEQ ID NO:1. These amino acids areresponsible for a distinct negative charge distribution on the surfaceof the C-terminal calcium-binding loop. It was shown that a peptideconsisting of the 12 amino acids forming the C-terminal calcium-bindingloop is immunologically active. Thus, the mutation of the abovementioned aspartic acid residues will heavily influence this epitope.Furthermore, at least one of the amino acids within the uncharged ridgemay be substituted or deleted. These are phenylalanine 57, asparagine58, isoleucine 61, serine 62, asparagine 65, alanine 66, proline 68 andmethionine 5 (which is provided from the opposite monomer chain) of SEQID NO:1.

It has further been found that recombinantly produced Phi p 7, whichcorresponds to naturally occurring Phi p 7, occurs as a novel dimerassembly adopting an extended conformation. Two protein monomersassemble in a head to tail arrangement with domain-swapped EF-handpairing. The intertwined dimer adopts a barrel-like structure with anextended hydrophobic cavity providing a ligand-binding site. Calciumbinding acts as a conformational switch between an open and a closeddimeric form of Phi p 7. Therefore, disruption of dimer assembly may beenvisaged according to the invention. This strategy targets the highcross-linking activity of Phi p 7 on the effector cell-bound IgEantibodies. As a result of the domain swapping, the protein assemblesinto a highly symmetric dimer. This leads to the doubling of (identical)IgE epitopes. A conversion of the domain-swapped dimer into thecorresponding monomer is expected to maintain the IgE epitopes but tostrongly diminish the allergy-eliciting cross-linking activity.Therefore, according to the invention mutations of the hinge loop thatdestabilize the extended linker region between the N— and C-terminalcalcium-binding domains is proposed. First mutation of at least one ofthe amino acids that provide the discrete hydrogen bonds and thusstabilize the rigid conformation of the linker region is suggested:these are the highly conserved residues arginine 30, glycine 33, serine34, threonine 35, serine 36 and glutamic acid 39 (of SEQ ID NO:1). Theexchange of these residues against amino acids that are unable to formhydrogen bonds (e.g. bulky non-polar amino acids) would strongly affectthe stability of the hinge-loop conformation. Furthermore we suggest thesubstitution of the hinge loop forming residues (especially glycine 33,serine 34, threonine 35, serine 36) by a stretch of small flexible aminoacids (such as glycine or alanine), either one by one or by introducinga longer stretch, elongating the hinge loop and simultaneouslyincreasing its flexibility. Another possibility is the exchange of theshort rigid Phi p 7 hinge loop (glycine 33, serine 34, threonine 35,serine 36) with corresponding loop-forming sequences known fromintra-domain paired EF-hand proteins. All these mutations enable theformation of a monomeric Phi p 7 with intramolecular EF-hand pairing.

Furthermore, the dimeric structure may be disrupted by mutation of atleast one of the hydrophobic residues that provide interaction betweenthe E-helices and the Z-helix of the opposite monomer in thedomain-swapped dimer. These amino acids are isoleucine 8, phenylalanine12 (part of the N-terminal E-helix), threonine 35, valine 40, methionine43 and isoleucine 47 (part of the C-terminal E-helix), as well as theresidues methionine 71, valine 74, alanine 75, lysine 76, valine 77 andphenylalanine 78 (part of the Z-helix) of SEQ ID NO:1. Mutation of theseamino acids (e.g. by charged or bulky residues) will stronglydestabilize the symmetric dimer assembly.

The polypeptides of the present invention have reduced allergenicactivity compared to wild type Phi p 7. According to the invention theallergenic activity of a sample is determined by determining the IgEantibodies which are induced in a test animal upon application of thesample. The allergenic activity is preferably defined in suitable invitro or in vivo tests. The allergenic activity may be determined in askin test as described in van Hage-Hamsten et al. J. Allergy Clin.Immunol. 1999, 104, pp. 969-977 or in Pauli et al. Clin. Exp. Allergy2000, 30, pp. 1076-1084. The allergenic activity of wild type Phi p 7may be determined using recombinantly produced Phi p 7 consisting of theamino acid sequence as shown in SEQ ID NO:1.

Preferably the allergenic activity of the polypeptide is less than 50%of the allergenic activity of the wild type Phi p 7. More preferably theallergenic activity of the polypeptide is less than 25% of the wild typeprotein. In the most preferred embodiment the polypeptide hassubstantially no allergenic activity. Generally, the histamine releaseinduced by the polypeptide of the invention is significantly reducedcompared to the histamine release induced by Phi p 7. A preferred invitro test for determining the histamine release is the basophilhistamine release assay as described in Vrtala et al., J. Clin. Invest.1997, 99, pp. 1673-1681. Preferably, the histamine release is reduced byat least 25%, more preferably by at least 50%, most preferably by atleast 75%, determined at that concentration of allergen at which Phi p 7shows maximum histamine release.

The polypeptides of the invention show reduced binding to IgE antibodiesfrom timothy grass pollen allergic patients compared with wild type Phip 7 (SEQ ID NO:1). The IgE binding activity is preferably reduced by atleast 25%, more preferably by at least 50%, most preferably by at least75%. Recombinant Phi p 7 consisting substantially of the amino acidsequence as shown in SEQ ID NO:1 can be used to determine the IgEbinding activity of wild type Phi p 7. IgE binding of polypeptides maybe determined by Western blot analysis or dot blot experiments usingserum from a timothy grass pollen allergic patient. Timothy grass pollenallergy is diagnosed according to a case history indicative for timothygrass pollen allergy (i.e., seasonal symptoms of allergy during theflowering period of grasses), positive skin test reaction to timothygrass pollen allergens and/or the detection of specific IgE antibodiesto timothy grass pollen allergens in serum. Dot blots can be quantifiedby measuring the amount of ¹²⁵I-labeled anti-human IgE antibodies bygamma counting as described (Niederberger et al. J. Allergy Clin.Immunol. 1998, 102, 579-591).

It has also been found that the Phi p 7-derived polypeptides of theinvention induce IgG antibody responses in vivo. Therefore, thepolypeptides described above comprise at least one IgG epitope. Apolypeptide comprises at least one IgG epitope when it is capable ofeliciting an IgG antibody response in an individual or a test animal. Acorresponding test for determining an IgG response is described inExample 3. More preferably, these IgG antibodies are “blockingantibodies” or “protective antibodies” which prevent IgE antibodies frombinding to Phi p 7. A significant reduction of allergic symptoms may beachieved in this way.

It has been found that the polypeptides of the invention which carrymutations corresponding to the mutations represented by amino acidsequences SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ IDNO:6 have unexpected advantageous properties. The amino acid positionstargeted in these amino acid sequences can be substituted or deleted inother 2-EF hand pollen allergens as well. Therefore, the presentinvention relates to a mutated polypeptide derived from a 2-EF handpollen allergen, wherein in respect to the wild type sequence of the2-EF hand pollen allergen amino acid positions have been substituted ordeleted which correspond to the amino acid residues which aresubstituted or deleted in respect to SEQ ID NO:1 in SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. The identification ofamino acid positions in other 2-EF hand pollen allergens whichcorrespond to the amino acid positions mutated in anyone of SEQ ID NO:2to 6 is within the level of ordinary skill. For example, the skilledperson may align anyone of the sequences as shown in SEQ ID NO:1 to 6with the amino acid sequence of a given 2-EF hand pollen allergen usingthe program Blast 2 sequences described above. From the alignment thecorresponding amino acid positions to be mutated can easily be derived.As an example, FIG. 9 shows an alignment of Phi p 7 and Aln g 4. Theamino acid positions to be substituted or deleted correspond to D17,E24, D52 and/or E59 of SEQ ID NO:1. These positions correspond to D24,E31, D59 and E66 of the amino acid sequence of Aln g 4 (SEQ ID NO:7).The polypeptides derived from 2-EF hand pollen allergens may becomprised in a larger polypeptide or be coupled to a suitable carrierprotein such as KLH. They also have reduced allergenic activity and IgEbinding capacity compared with their respective wild type forms and arecapable of inducing an IgG response as described supra. The amino acidsequences of several 2-EF hand pollen allergens are known:

Aln g 4 (alder) (Hayek et al. J. Immunol. 1998, 161, 7031-7039)

Cyn d 7 (Bermuda grass) (Suphioglu et al. FEBS Lett. 1997, 402,167-172)

Bra r 1 (rape-Brassica) (Toriyama et al. FEBS Lett. 1998, 424, 234-238)

Bet v 4 (birch) (Twardosz et al. Biochem. Biophys. Res. Commun. 1997,239,197-204)

Ole e 3 (olive) (Ledesma et al. Eur. J. Biochem. 1998, 258, 454-459)

The amino acid sequences of Aln g 4, Cyn d 7, Ole e 3, Bet v 4 and Bra r1 are shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 andSEQ ID NO:11, respectively. The amino acid at position 71 of SEQ IDNO:10 may also be alanine.

It is to be noted that polypeptides derived from 2-EF hand pollenallergens other than Phi p 7 are only claimed as far as they comprisesubstitutions or deletions at the same amino acid positions as SEQ IDNO:2 to 6.

The invention relates to a mutated polypeptide derived from Aln g 4,wherein in respect to the amino acid sequence as shown in SEQ ID NO:7the amino acids at position 31 and 66, preferably at position 24, 31 and66, more preferably at position 24, 31, 59 and 66 are substituted. Theinvention further relates to a polypeptide consisting substantially ofamino acids 2 to 44 or 43 to 85 of SEQ ID NO:7, optionally coupled to acarrier molecule such as KLH.

Another aspect of the invention is a mutated polypeptide derived fromCyn d 7, wherein in respect to the amino acid sequence as shown in SEQID NO:8 the amino acids at position 26 and 61, preferably at position19, 26 and 61, more preferably at position 19, 26, 54 and 61 aresubstituted. Another aspect is a polypeptide consisting substantially ofamino acids 2 to 39 or 38 to 80 of the amino acid sequence as shown inSEQ ID NO:8, optionally coupled to a carrier molecule such as KLH.

In yet another aspect the invention relates to a mutated polypeptidederived from Ole e 3, wherein in respect to the amino acid sequence asshown in SEQ ID NO:9 the amino acids at position 30 and 65, preferablyat position 23, 30 and 65, more preferably at position 23, 30, 58 and 65are substituted. The invention also relates to a polypeptide consistingsubstantially of amino acids 2 to 43 or 42 to 84 of the amino acidsequence as shown in SEQ ID NO:9, optionally coupled to a carriermolecule such as KLH.

In yet another aspect the invention relates to a mutated polypeptidederived from Bet v 4, wherein in respect to the amino acid sequence asshown in SEQ ID NO:10 the amino acids at position 31 and 66, preferablyat position 24, 31 and 66, more preferably at position 24, 31, 59 and 66are substituted. The invention also relates to a polypeptide consistingsubstantially of amino acids 2 to 44 or 43 to 85 of the amino acidsequence as shown in SEQ ID NO:10, optionally coupled to a carriermolecule such as KLH.

In yet another aspect the invention relates to a mutated polypeptidederived from Bra r 1, wherein in respect to the amino acid sequence asshown in SEQ ID NO:11 the amino acids at position 25 and 60, preferablyat position 18, 25 and 60, more preferably at position 18, 25, 53 and 60are substituted. The invention also relates to a polypeptide consistingsubstantially of amino acids 2 to 38 or 37 to 79 of the amino acidsequence as shown in SEQ ID NO:11, optionally coupled to a carriermolecule such as KLH.

Wild type Phi p 7 represented by the amino acid sequence as shown in SEQID NO:1 and proteins comprising wild type Phi p 7 are not within thescope of the present invention. Naturally occurring 2-EF hand pollenallergens or recombinant proteins consisting of the same amino acids arenot claimed in this application (e.g. Bet v 4, Bra r 1, Aln g 4, Cyn d7, etc.).

The present invention also provides polynucleotide molecules, includingDNA and RNA molecules, that encode the polypeptides disclosed herein.Those skilled in the art will readily recognize that, in view of thedegeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules. The polynucleotide may besingle or double-stranded. It is to be recognized that according to thepresent invention, when a polynucleotide is claimed as described herein,it is understood that what is claimed are both the sense strand, theantisense strand and the DNA as double-stranded having both the senseand antisense strand annealed together by their respective hydrogenbonds. Also claimed is the messenger RNA (mRNA) which encodes thepolypeptides of the present invention. Messenger RNA will encode apolypeptide using the same codons as those used by DNA, with theexception that each thymine nucleotide (T) is replaced by a uracilnucleotide (U).

Methods for preparing DNA and RNA are well known in the art. Afull-length clone encoding Phi p 7 can be obtained by conventionalcloning procedures. The DNA encoding Phi p 7 may be amplified bypolymerase chain reaction (PCR) employing suitable specific primers. Thepolynucleotides of the present invention may also be synthesizedchemically, for example using the phosphoramidite method.

The invention further relates to a vector or plasmid containing apolynucleotide as described above. In general, the polynucleotidesequence encoding a polypeptide of the invention is operably linked toother genetic elements required for its expression, generally includinga transcription promoter and terminator, within an expression vector.The vector will also commonly contain one or more selectable markers orone or more origins of replication, although those skilled in the artwill recognize that within certain systems selectable markers may beprovided on separate vectors, and replication of the exogenous DNA maybe provided by integration into the host cell genome. Selection ofpromoters, terminators, selectable markers, vectors and other elementsis a matter of routine design within the level of ordinary skill in theart. Many such elements are described in the literature and areavailable through commercial suppliers.

Another aspect of the invention is a host cell transformed ortransfected with a vector or a plasmid according to the invention. Thehost cells may be prokaryotic or eukaryotic cells. Prokaryotic hostcells, including strains of the bacteria Escherichia coli, Bacillus andother genera are useful host cells within the present invention.Techniques for transforming these hosts and expressing foreign DNAsequences cloned therein are well known in the art (see, e.g., Sambrooket al., Molecular Cloning, a Laboratory Manual, Cold Spring HarborLaboratory, 1989).

The host cells of the invention may be used to produce the polypeptidesof the invention. Yet another aspect of the invention therefore is amethod of preparing a polypeptide according to the invention comprisingculturing host cells described above under conditions that saidpolypeptide is expressed and optionally recovering said polypeptide fromthe host cells. When expressing a polypeptide of the invention inbacteria such as E. coli, the polypeptide may be retained in thecytoplasm, possibly as insoluble inclusion bodies. In this case, thecells are lysed and the inclusion bodies are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded by diluting the denaturant, such as bydialysis against a solution of urea and a combination of reduced andoxidized glutathione, followed by dialysis against a buffered salinesolution.

Transformed or transfected host cells are cultured according toconventional procedures in culture medium containing nutrients and othercomponents required for the growth of the chosen host cells. A varietyof suitable media, including defined media and complex media, are knownin the art and generally include a carbon source, a nitrogen source andminerals. It is preferred to purify the peptides of the presentinvention to ≧80% purity, more preferably ≧95% purity, and particularlypreferred is a pharmaceutically pure state that is greater than 99.9%pure with respect to contaminating macromolecules, particularly otherproteins and nucleic acids, and free of infectious and pyrogenic agents.Preferably, a purified polypeptide of the invention is substantiallyfree of other polypeptides. Expressed recombinant polypeptide of theinvention can be purified using fractionation and/or conventionalpurification methods and media. Ammonium sulfate precipitation and acidor chaotrop extraction may be used for fractionation of samples. Thepolypeptides of the invention may also be isolated by affinitychromatography using antibodies directed to the polypeptide. Shorterpolypeptides are preferably purified using HPLC. Methods of proteinpurification are described e.g. in Methods in Enzymology, Volume 182.Guide to Protein Purification. Academic Press New York 1990 and Scopes,Protein Purification. Springer Verlag, Heidelberg 1994.

The polypeptides of the invention may also be prepared through chemicalsynthesis, for example, as described by Merryfield, J. Am. Chem. Soc.85:2149, 1963 and Etherton et al., Solid Phase Peptide Synthesis: APractical Approach, IRL Press, Oxford 1989.

The invention further relates to the use of the polypeptide of theinvention for the manufacture of a medicament for treating and/orpreventing an allergic disorder. The disorder usually is an allergy toone or more 2-EF hand pollen allergens, e.g. to Phi p 7. It has beenfound that Phi p 7 contains most of the relevant IgE epitopes of thefamily of 2-EF hand pollen allergens. Therefore, the polypeptides may beused in the treatment of allergies to almost any 2-EF hand pollenallergen. Preferably, the allergic disorder to be treated is allergy toat least one of the proteins Bet v 4, Bra r 1, Aln g 4, Bra n 1, Cyn d7, Ole e 3, Syr v 3 and/or Phi p 7. The medicament may be used for thetherapeutic treatment of an allergic disorder or for prophylacticvaccination to prevent development of the disorder.

The invention also pertains to a pharmaceutical composition comprisingat least one polypeptide of the invention. The composition may furthercomprise a pharmaceutically acceptable carrier or diluent. Preferably,the polypeptide of the invention has been coupled to a carrier moleculesuch as KLH.

Another aspect of the invention is a pharmaceutical kit comprising atleast one polypeptide of the invention. The kit may comprise two or moredifferent polypeptides according to the present invention. In oneembodiment the kit comprises at least one mutated polypeptide derivedfrom Phi p 7 and at least one mutated polypeptide derived from another2-EF hand pollen allergen. Other allergens from timothy grass pollen orfrom other pollen and their epitopes may be contained. In anotheraspect, the mutated polypeptide derived from Phi p 7 may be onecomponent in a prophylactic pharmaceutical composition for vaccinationcontaining the most important allergens from different allergen sources(mite, cat, pollen, mushrooms, etc.).

For pharmaceutical use, the polypeptides of the present invention areformulated for oral or parenteral, particularly subcutaneous, deliveryaccording to conventional methods. In general, pharmaceuticalformulations will include a polypeptide of the invention in combinationwith a pharmaceutically acceptable vehicle, such as saline, bufferedsaline, 5% dextrose in water or the like. Formulations may furtherinclude one or more excipients, preservatives, solubilizers, bufferingagents, albumin to prevent protein loss on vial surfaces, etc. Methodsof formulation are well known in the art and are disclosed, for example,in Remington: The Science and Practice of Pharmacy, Gennaro, Ed., MackPublishing Co., Easton, Pa., 19th Edition 1995. Therapeutic orprophylactic doses will generally be in the range of 0.1-100 μg perinjection in a volume of 100-200 μl, with the exact dose determined bythe physician according to accepted standards, taking into account thenature and severity of the condition to be treated, patient traits, etc.Determination of dose is within the level of ordinary skill in the art.The amount may vary depending on the mode of treatment. Duringimmunotherapy treatment single doses of about 25 μg to 75 μg can beadministered in a volume of about 100 μl per injection. In case of oraladministration a dosage of 0.1 μg to 50 mg can be envisaged. In the caseof vaccinations, patients are usually not treated several times a dayexcept for “rush immunotherapy”. Common immunotherapies includeapproximately 8 preseasonal vaccinations that are administered inintervals of one to two weeks and that are continued over a period of 2to 3 years. Preferably, 4 injections per year with an interval of 3months over 3 to 5 years are applied. In a particular embodiment, morethan one polypeptide is contained in the pharmaceutical composition.

DESCRIPTION OF THE TABLES AND FIGURES

FIG. 1. CD spectra of Phi p 7, the Phi p 7-derived peptides and the Phip 7-derived mutants (M1.6, M2A, M4).

FIG. 2A, B. Reduction of the IgE binding capacity of Phi p 7-derivedmutants and of Phi p 7-derived peptides.

Nitrocellulose blotted rPhi p 7 (lane Phi p 7) and rPhi p 7-derivedmutants (lanes M1.6, M2A, M4) were probed with serum from a Phi p7-sensitized grass pollen allergic patient. Bound IgE antibodies weredetected with ¹²⁵I-labeled anti human IgE antibodies (FIG. 2A).Nitrocellulose dotted Phi p 7 and Phi p 7-derived mutants (M1.6, M2A,M4) as well the Phi p 7-derived peptides were probed with sera from tenPhi p 7-sensitized grass pollen allergic patients. Bound IgE antibodieswere detected with ¹²⁵I-labeled anti human IgE antibodies (FIG. 2B).

FIG. 3A, B. Induction of histamine release from basophils of a timothygrass pollen allergic patient. Granulocytes of a timothy grass pollenallergic patient were incubated with various concentrations (x-axis) ofrPhi p 7 wild type (P7), Phi p 7-derived peptides (P1, P2), Phi p7-derived mutant (M4) or another antigen (Hom s 4) (FIG. 3A).Granulocytes of the same patient were also incubated with variousconcentrations (x-axis) of KLH-coupled rPhi p 7 wild type (P7-K) , Phi p7-derived peptides (P1-K, P2-K) or Phi p 7-derived mutant (M4-K) (FIG.3B).The percentage of histamine released into the cell-free culturesupernatant is displayed on the y-axis.

FIG. 4. Rabbit antisera against KLH-coupled peptides or against coupledand uncoupled Phi p 7-mutant react with complete Phi p 7 wild type.Nitrocellulose-dotted Phi p 7 and Phi p 7-derived peptides or mutantwere probed with the corresponding rabbit antiserum in differentconcentrations (1:500, 1:1000, 1:2000). Bound rabbit antibodies weredetected with ¹²⁵I-donkey anti rabbit antibodies.

FIG. 5. Rabbit antisera against KLH-coupled peptides or against coupledand uncoupled Phi p 7-mutant react with a Phi p 7-cross-reactivecalcium-binding protein from alder pollen, Aln g 4.

Nitrocellulose dotted Aln g 4 was probed with rabbit antiserum (anti-M4,anti-M4-KLH, anti-P1-KLH, anti-P2-KLH). Bound rabbit antibodies weredetected with ¹²⁵I-donkey anti-rabbit antibodies.

FIG. 6 (Table 1). Characteristics of two non-allergenic Phi p 7-derivedsynthetic peptides and three Phi p 7-derived mutants with reducedallergenicity. Position, sequence, length, molecular weight, isoelectricpoint and fold are displayed. Mutated amino acids are printed in boldletters.

FIG. 7 (Table 2). Immediate type skin reactions to complete rPhi p 7,Phi p 7-derived peptides and to a Phi p 7-derived mutant (M4). Threetimothy grass pollen allergic patients (#1-3) and a non-allergicindividual (#4) were tested. The mean wheal diameters (mm) are displayedfor two different concentrations of rPhi p 7, for timothy grass pollenextract, histamine and two different concentrations of the two peptidesand the mutant.

FIG. 8 (Table 3). Inhibition of rabbit anti Phi p 7 peptide and ofrabbit anti Phi p 7 mutant antisera to serum IgE binding of grass pollenallergic patients to rPhi p 7.

FIG. 9. Alignment of the amino acid sequences of Phi p 7 and Aln g 4.Amino acid residues which may preferably be substituted or deleted in2-EF hand pollen allergens are shown in bold.

The invention is further illustrated by the following non-limitingexamples:

EXAMPLE 1

Characterization of two peptides comprising the complete N-terminal orthe C-terminal calcium-binding domain of Phi p 7 and of Phi p 7-derivedmutants with amino acid exchanges in the first and in the secondEF-hand.

Peptide Synthesis:

Peptides were synthesized using Fmoc(9-fluorenylmethoxycarbonyl)-strategy with HBTU(2-(1H-benzotriazoI-1-yl) 1,1,3,3 tetramethyluroniumhexafluorophosphat)-activation (0.1 mmol small-scale cycles) on theApplied Biosystems peptide synthesizer Model 433A (foster City, Calif.).preloaded PEG-PS (polyethyleneglycol polysterene) resins (0.15-0.2mmol/g loading) (per Septive Biosystems, Warrington, UK) were used assolid phase to build up the peptides. Chemicals were purchased fromApplied Biosystems. Coupling of amino acids was confirmed byconductivity monitoring in a feedback control system. One cysteineresidue was added to each peptide to facilitate coupling of the peptidesto carriers. Peptides were cleaved from the resins with a mixture of:250 μl dest. Water, 250 μl Triisopropylsilan (Fluka, Buchs,Switzerland), 9.5 ml TFA for 2 h and precipitated intert-Butylmethylether (Fluka, Buchs, Switzerland). The identity of thepeptides was checked by mass-spectrometry and they were purified to >90%purity by preparative HPLC (PiChem, Graz, Austria).

Generation, Expression and Purification of a Phi p 7-Derived Mutants:

Point mutations were introduced into the cDNA of Phi p 7, cloned intothe expression vector pET17b, using a Chameleon double-strandedsite-directed mutagenesis kit (Stratagene, East Kew, Australia).Specific primers were designed to mutate Phi p 7 at specific sites. Thefollowing mutations were made: Mutant 1.6 (SEQ ID NO:4): 24E→24A (firstEF-hand); 59E→59A (second EF-hand). Mutant 2A (SEQ ID NO:5): 17D→A,24E→24A (first EF-hand); 59E→59A (second EF-hand). Mutant 4 (SEQ IDNO:6): 17D→A, 24E→24A (first EF-hand); 52D→52A, 59E→59A (secondEF-hand). The recombinant Phi p 7-derived mutants were expressed inEscherichia coli BL21 (DE3). E. coli were grown to an OD₆₀₀ of 0.4 inLB-medium containing 100 mg/l ampicillin. The expression of recombinantproteins was induced by adding isopropyl-β-thiogalactopyranoside to afinal concentration of 1 mM and culturing for additional 4 hours at 37°C. E. coli cells from a 500 ml culture were harvested by centrifugation,resuspended in 10 ml PBS and homogenized using an ultraturrax (Ika,Heidelberg, Germany). A fraction containing soluble proteins wasobtained after centrifugation of the homogenate at 10.000 rpm (Sorval,RC5C, SS34 rotor) for 30 min at 4° C. Enrichment of the protein in thesoluble fraction and removal of contaminating proteins were achieved byaddition of 70% w/v ammonsulfate to the soluble E.coli fraction andcentrifugation (18.000 rpm, Sorval SS34, 4° C., 30 min.). The solublePhi p 7-derived mutant fraction was dialyzed against water, lyophilized,resuspended in 50 ml buffer A (25 mM Imidazole, 1 mM β-mercaptoethanol,pH=7.4), and applied to a DEAE anion exchange column (Pharmacia,Uppsala, Sweden). The mutant was eluted by a NaCl gradient (buffer Acontaining 500 mM NaCl) at ˜200 mM NaCl. Fractions containing pure Phi p7-derived mutant were pooled, dialyzed against water, and lyophilized.Protein samples were analyzed for purity by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteinstaining.

Coupling of Peptides and Mutant (M4) to KLH

HPLC-purified peptides or Phi p 7-derived mutant as well as Phi p 7 wildtype were coupled to KLH (keyhole limpet hemocyanin, MW 4.5×10³−1.3×10⁷,Pierce, Rockford, Ill.) according to the manufacturers advice andpurified using a Conjugation kit (Sigma, St. Louis).

Secondary structure analysis (CD)

CD measurements were carried out on a Jasco J-715 spectropolarimeterusing a 0.1 cm pathlength cell equilibrated at 20° C. Spectra wererecorded with 0.5 nm resolution at a scan speed of 100 nm/min andresulted from averaging 3 scans. The final spectra werebaseline-corrected by subtracting the corresponding MilliQ spectraobtained under identical conditions. Results were fitted with thesecondary structure estimation program J-700.

The far-UV spectra indicate, that the point mutants contain considerableamount of α-helical structure. The spectra are characterized by minimaat 224 and 208 nm and a strong maximum below 200 nm. The a-helicalcontent is identical for the various point mutations. The nativerecombinant Phi p 7 showed a significant larger α-helical signal. Asecond analysis comparing Phi p 7 and point mutant-4 revealed, that—incontrast to the normalized spectrum of rPhi p 7 the signal for mutant-4significantly decreased by approximately 20%. Furthermore the minimum at208 nm is slightly shifted to a smaller wavelength and the zero-crossingof the curve is below 200 nm. These findings are indicative for anincreasing portion of random-coil secondary structure within themutant-4 protein.

The N-terminal peptide exhibited a random coil secondary structure, withcharacteristic minima at 200 and 225 nm. This points to the fact, thattruncation of intact rPhi p 7 protein not only leads to the disruptionof the overall assembly, but furthermore affects the fold of the singleEF-hand domain. This could also be seen for the corresponding C-terminalpeptide, as this protein was not only unfolded, but furthermoredisplayed a significant decreased solubility and precipitation.

EXAMPLE 2

Fragmentation of Phi p 7 at specific sites leads to loss of IgE bindingcapacity, mutation of Phi p 7 at specific sites of the calcium-bindingdomains leads to decreased IgE binding capacity.

a) The Phi p 7-Derived Mutants and Phi p 7-Derived Peptides Show ReducedIgE Binding:

The IgE binding capacity of purified Phi p 7-derived mutants (M1.6, M2A,M4) was compared with that of Phi p 7 wild type by western blot analysisusing serum from a timothy grass pollen allergic patient (FIG. 2A) aswell as by dot blot experiments using sera from ten timothy grass pollenallergic patients (FIG. 2B). The western blot analysis showed a reducedIgE binding capacity for all mutants compared to Phi p 7. The strongestreduction was detected for the mutant M4 (FIG. 2A). This finding wasconfirmed by the dot blot experiments: only four out of ten patientsshowed weak IgE binding to M4, five out of ten reacted with M2A, six outof ten with M1.6 (FIG. 2B). The double mutant (M4) hence exhibited thestrongest reduction of IgE binding capacity.

The IgE binding capacity of Phi p 7-derived peptides was compared withthat of Phi p 7 wild type by dot blot experiments using sera from tentimothy grass pollen allergic patients (FIG. 2B). For both peptides theIgE binding capacity was completely abolished, when tested with serumfrom ten timothy grass pollen allergic patients.

b) Reduced basophil histamine Release by Phi p 7 Mutant and Peptides

Next the Phi p 7-derived peptides and the Phi p 7-derived mutant werecompared with Phi p 7 wild type for their capacity to induce histaminerelease from basophils of a timothy grass pollen allergic individual.

Granulocytes were isolated from heparinized blood samples of a timothygrass pollen allergic individual by dextran sedimentation. Cells wereincubated with increasing concentrations (10⁻⁶-10 μg/ml) of each peptideor the mutant, and, for control purposes, with rPhi p 7 wild type.Histamine released in the cell-free culture supernatant was measured byradioimmunoassay (Immunotech, Marseille, France). Total histamine wasdetermined after freezing and thawing of the cells. Results aredisplayed as mean values of triplicate determinations.

As exemplified in FIG. 3 it was found that none of the peptides (coupledor uncoupled) induced histamine release up to an concentration of 10⁻³μg/ml. The Phi p 7-derived mutant M4 induced a dose-dependent release ofhistamine with a maximum release at a concentration of 10⁻³ μg/ml,whereas Phi p 7 wild type induced a maximum histamine release already ata concentration at 10⁻⁴ μg/ml. Coupling to KLH did not increase theallergenic activity of the mutant and peptides (FIG. 3B).

c) Reduced Allergenic in vivo Activity of the Mutant and the Peptides

In vivo testing in timothy grass pollen allergic patients confirmed theabolished allergenic activity of the Phi p 7-derived peptides and thereduced allergenicity of the Phi p 7-derived mutant M4.

The in vivo allergenic activity of the peptides and the mutant wasstudied by skin prick testing (SPT) in 3 timothy grass pollen allergicpatients and a non-atopic individual. SPTs were performed on theindividuals' forearms. Twenty microliter aliquots containing 2concentrations of complete rPhi p 7 or Phi p 7-derived mutant M4 (2μg/ml, 8 μg/ml) as well as two concentrations of Phi p 7-derivedpeptides (1 μg/ml m, 4 μg/ml ) were applied. In addition, standardizedskin prick solutions (timothy grass pollen extract and histamine)(Allergopharma, Reinbeck, Germany) were tested. Reactions were recorded20 minutes after SPT by photography and by transferring the ballpointpen-surrounded wheal area with a scotch tape to paper. The mean whealdiameter (Dm) was calculated by measuring the maximal longitudinal andtransversal diameter and dividing their sum by 2.

Skin prick tests were performed in 3 timothy grass pollen allergicpatients and a non-allergic individual with rPhi p 7, Phi p 7-derivedpeptides and a Phi p 7-derived mutant M4 (Table 3). None of the peptidesinduced any immediate skin reactions when applied at a concentration of4 μg/ml, whereas Phi p 7 wild type already induced immediate type skinreactions at a concentration of 2 μg/ml. M4 induced immediate skinreactions that were moderately weaker than those induced by Phi p 7 wildtype (mean wheal diameter induced by Phi p 7 at a concentration of 8μg/ml: 9.3 mm; mean wheal diameter induced by M4 at a concentration of 8μg/ml: 7 mm). All timothy grass pollen allergic patients displayedimmediate skin reactions to timothy grass pollen extract. Thenon-allergic individual showed no reactions to timothy grass pollenextract, rPhi p 7, the Phi p 7-derived peptides or the Phi p 7-derivedmutant (Table 3:#4). All individuals reacted after testing withhistamine, used as a positive control (Table 3).

EXAMPLE 3

Immunization with Phi p 7-derived peptides and the Phi p 7-derivedmutant induces IgG antibodies that recognize rPhi p 7 wild type as wellas a cross-reactive allergen from alder, Aln g 4.

In order to test whether immunization with Phi p 7-derived peptides orPhi p 7-derived mutant M4 will induce IgG antibodies that react withcomplete Phi p 7 molecule and Phi p 7 cross-reactive allergens, rabbitswere immunized with Phi p 7 wild type, the peptides or the Phi p7-mutant as well as with KLH-conjugated peptides/proteins using Freund'sadjuvant. Eight rabbits were immunized with a peptide-KLH conjugate, themutant-KLH conjugat, Phi p 7-KLH conjugate, unconjugated peptides,mutant and Phi p 7 wild type, respectively, (200 μg/injection) usingFreund's complete and incomplete adjuvants (Charles River, Kibllegg,Germany). Serum samples were obtained in four week intervals. Sera werestored at −20° C. until analysis.

Reactivity of peptide-induced IgG antibodies and Phi p 7-mutant-inducedIgG antibodies to rPhi p 7 wild type and a cross reactive allergen wasstudied by dot blot experiments. Phi p 7 wild type as well as thecorresponding immunogen (peptide 1, peptide 2, Mut-4) were dotted ontonitrocellulose-strips (1 μg/dot). Strips were exposed to differentdilutions of rabbit antiserum (1:500, 1:1000, 1:2000).

Similarly, recombinant Aln g 4, a Phi p 7-cross-reactive calcium-bindingallergen from alder, was dotted onto nitrocellulose and strips wereexposed to 1:1000 diluted rabbit antiserum. Bound rabbit antibodies weredetected with a 1:1000 diluted ¹²⁵I-labeled donkey anti-rabbit antiserum(Amersham Pharmacia Biotech).

The peptides coupled to KLH induced IgG anti-Phi p 7 antibody responses(FIG. 4), as well as the Phi p 7-derived mutant (M4, coupled anduncoupled). Similarly, rabbit antisera raised against M4, M4-KLH and thecoupled peptides recognized the Phi p 7-cross-reactive allergen, Aln g 4(FIG. 5).

EXAMPLE 4

Anti-peptide and anti-mutant antisera inhibit the binding of serum IgEfrom grass pollen allergic patients to complete rPhi p 7.

The capacity of anti-Phi p 7 peptide and anti-Phi p 7 mutant antibodiesto inhibit the binding of allergic patients' serum IgE to complete rPhip 7 was studied by ELISA competition using sera from four grass pollenallergic patients (Table 3).

The ability of peptide or mutant-induced rabbit IgG to inhibit thebinding of allergic patients' IgE to complete Phi p 7 was investigatedby ELISA competition assay. ELISA plates (Nunc Maxisorp, Rokslide,Denmark) were coated with rPhi p 7 (1 μg/ml) and preincubated eitherwith a 1:250 dilution of each of the anti-peptide antisera (anti-P1-KLH,anti-P2-KLH), the anti-mutant antiserum (anti-M 4-KLH) and, for controlpurposes, with the corresponding preimmunserum. After washing plateswere incubated with 1:3 diluted sera from four Phi p 7-sensitized grasspollen allergic patients and bound IgE antibodies were detected with amonoclonal rat anti-human IgE antibody (Pharmingen, San Diego, Calif.),diluted 1:1000, followed by a 1:2000 diluted HRP-coupled sheep anti ratIg antiserum (Amersham Pharmacia Biotech, Uppsala, Sweden). Thepercentage inhibition of IgE binding achieved by preincubation with theanti-peptide or anti-mutant antisera was calculated as follows: %inhibition of IgE binding=100-OD_(l)/OD_(P)×100. OD_(l) and OD_(P)represent the extinctions after preincubation with the rabbits immuneand preimmune serum, respectively.

Strongest inhibition of IgE binding was observed after preincubationwith anti-peptide 2 (47% average inhibition). Anti-mutant-KLH coupledantibodies exhibited a lower capacity to inhibit serum IgE binding toPhi p 7 (23.4% average inhibition). Inhibition of serum IgE bindingafter preincubation with anti-mutant could be detected in two out offour sera (average inhibition 5.6%) and in only one out of four seraafter preincubation with anti-peptide 1.

1. A mutated polypeptide derived from the pollen allergen Phi p 7, saidpolypeptide selected from the group consisting of the sequences as shownin SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6,whereby said polypeptide has a reduced allergenic activity compared towild type Phi p
 7. 2. A polypeptide according to claim 1 which iscapable of inducing an IgG response in a mammal.
 3. A polypeptideaccording to claim 1 or 2 which induces a histamine release which issignificantly reduced compared with wild type Phi p
 7. 4. Apolynucleotide encoding a polypeptide according to claim
 1. 5. A vectoror plasmid containing a polynucleotide according to claim
 4. 6. A hostcell transformed or transfected with a vector or a plasmid according toclaim
 5. 7. A method of preparing a polypeptide comprising culturinghost cells according to claim 6 under conditions that said polypeptideis expressed and optionally recovering said polypeptide from said hostcells.
 8. A method of preparing a polypeptide according to claim 1comprising chemically synthesizing said polypeptide. 9-13. (canceled)14. A pharmaceutical composition comprising a polypeptide according toclaim 1 and a pharmaceutically acceptable carrier or diluent.
 15. A kitcomprising a polypeptide according to claim 1 and a pharmaceuticallyacceptable carrier or diluent.
 16. A method of treating and/orpreventing an allergic disorder, said method comprising administering toa patient in need thereof an effective amount of the polypeptide ofclaim
 1. 17. The method of claim 16, wherein said allergic disorder isan allergy to a two-EF hand pollen allergen.
 18. The method of claim 17,wherein said disorder is allergy to Phi p
 7. 19. The method of claim 17,wherein the allergic disorder is allergy to an allergen selected fromthe group consisting of Bet v 4, Bra r 1, Aln g 4, Bra n 1, Cyn d 7, Olee 3, Syr v 3 and/or Phi p 7.